Figure 3.

In Vitro Differentiation of Endoderm-Derived Hepatic Organoids into Mature Hepatocytes

(A) Confocal images of organoids cultured for 10–14 days in DM conditions stained for CK18, E-cadherin, A1AT, ZO1, and ALB. Nuclei were stained with DAPI. In the dataset for p10 organoids, CK18, E-CAD/A1AT, and ZO-1/ALB were stained in the whole-mount organoids. The others were stained from frozen sections.

(B) Immunohistochemical staining of organoids for ALB, CK19, and E-cadherin.

(C) Transmission electron microscopy image of an endoderm-derived hepatic organoid (eHEPO). Arrow shows apoptotic and multivesicular bodies (upper panel). White circles and arrow indicate intercellular junctional complexes (JC) and apical villus (AV), respectively (lower panel).

(D) Lentiviral albumin promoter-GFP reporter to monitor albumin expression in organoids. Representative bright-field and fluorescence microscopy images of pALB-GFP reporter bearing iPSCs at the indicated stages of differentiation. Flow-cytometry analysis to quantify ALB⁺ cells within organoid.

(E) GSEA analysis of differentially regulated genes in DM versus EM conditions. NES and FDR q value are listed for the liver-specific gene set analyzed.

(F) Heatmap showing the expression of EM- and DM-related genes.

(G) qPCR-based mRNA expression analysis of indicated genes in EM and DM organoids as well as human liver tissue. Fold changes were calculated as DM/EM and/or tissue/EM (n = 4 for each of three separate differentiation) (^∗p ≤ 0.05).